The Role of Cocaine- and Amphetamine-Regulated Transcript (CART) in Cancer: A Systematic Review

The functions of cocaine- and amphetamine-regulated transcript (CART) neuropeptide encoded by the CARTPT gene vary from modifying behavior and pain sensitivity to being an antioxidant. Putative CART peptide receptor GPR160 was implicated recently in the pathogenesis of cancer. However, the exact role of CART protein in the development of neoplasms remains unclear. This systematic review includes articles retrieved from the Scopus, PubMed, Web of Science and Medline Complete databases. Nineteen publications that met the inclusion criteria and describe the association of CART and cancer were analyzed. CART is expressed in various types of cancer, e.g., in breast cancer and neuroendocrine tumors (NETs). The role of CART as a potential biomarker in breast cancer, stomach adenocarcinoma, glioma and some types of NETs was suggested. In various cancer cell lines, CARTPT acts an oncogene, enhancing cellular survival by the activation of the ERK pathway, the stimulation of other pro-survival molecules, the inhibition of apoptosis or the increase in cyclin D1 levels. In breast cancer, CART was reported to protect tumor cells from tamoxifen-mediated death. Taken together, these data support the role of CART activity in the pathogenesis of cancer, thus opening new diagnostic and therapeutic approaches in neoplastic disorders.


Introduction
Cocaine-and amphetamine-regulated transcript (CART) was discovered for the first time in 1995, when a previously uncharacterized mRNA was identified in the rat stratum [1]. Its relative expression levels were prominently increased by acute cocaine or amphetamine administration, hence the name CART [1,2]. Soon, it was clear that in different species including humans, CART peptide is widely distributed over multiple areas of the brain, including, e.g., the pituitary gland, hypothalamus, brain stem or dorsal horns of the spinal cord [3,4]. It is also present in neuroendocrine cells of the intestine [5], pancreas [6] and adrenal medulla [3], as well as in the gastrin-producing G-cells in the stomach [7].
CART peptide originates from CART prepropeptide (CARTPT) encoded by the CARTPT gene. It is a conserved gene with high interspecies homology [8]. There are three distinct exons and two introns of the CARTPT gene [8]. In humans, it is located on chromosome 5q13-14 [9]. The single primary CARTPT mRNA codes for two CARTPT molecules-129 amino acids (aa) and 116 aa, generated due to alternative splicing [1] (Figure 1). Their N-terminal hydrophobic signal sequence (27aa) is later deleted, leaving two pro-CART isoforms-long (102 aa) and short (89 aa). Only the short form is expressed in humans [10]. Pro-CART proteins are processed by prohormone convertases during their transport from The most prominent effects of CART are related to modulation of behavior and brain function. CART peptide inhibits food intake, acts as a satiety signal and leads to weight loss in rats, but surprisingly, it can also be found in orexigenic neurons [12]. The anorexigenic activity of CART is modulated by leptin and neuropeptide Y [13]. CART also participates in stress modulation [14] and was implicated in the pathogenesis of psychiatric disorders such as depression or anxiety [15,16]. It may be also involved in the development of drug-dependency and addictions [17]. CART seems to be connected with hypersensitivity and neuropathic pain [18,19]. Furthermore, CART protein was reported to act as a strong antioxidant, neutralizing reactive oxygen species (ROS) [20]. ROS are able to damage cellular proteins, lipids and nucleic acids such as mitochondrial DNA (mtDNA). Thus, CART's localization in mitochondria may have protective effects [20]. CART55-102 The most prominent effects of CART are related to modulation of behavior and brain function. CART peptide inhibits food intake, acts as a satiety signal and leads to weight loss in rats, but surprisingly, it can also be found in orexigenic neurons [12]. The anorexigenic activity of CART is modulated by leptin and neuropeptide Y [13]. CART also participates in stress modulation [14] and was implicated in the pathogenesis of psychiatric disorders such as depression or anxiety [15,16]. It may be also involved in the development of drug-dependency and addictions [17]. CART seems to be connected with hypersensitivity and neuropathic pain [18,19]. Furthermore, CART protein was reported to act as a strong antioxidant, neutralizing reactive oxygen species (ROS) [20]. ROS are able to damage cellular proteins, lipids and nucleic acids such as mitochondrial DNA (mtDNA). Thus, CART's localization in mitochondria may have protective effects [20]. CART  also increases the function of mitochondrial respiratory chain complex II and depletes the levels of ROS in neurons after oxygen-glucose deprivation [21].
Putative CART receptors were identified recently. CART was suggested to be a ligand of G-protein-coupled receptor 160 (GPR160), previously known as one of the orphan Gprotein-coupled receptors [18]. Accumulating evidence suggests the association of GPR160 with cancer. In prostate cancer cells, the GPR160 knockdown increased expression of caspase 1 and interleukin 6, inhibited cell proliferation and evoked apoptosis [22]. Similarly, knockdown of GPR160 in glioma cells resulted in the promotion of apoptosis, decreased proliferation rate, reduced cell viability and diminished invasion ability [23]. In triplenegative breast cancer, GPR160 was found to be downregulated, and its higher level was correlated with better prognosis [24]. In contrast, amplification of GPR160 is connected with the development of nasopharyngeal carcinoma [25]. Additionally, in melanoma metastases, GPR160 expression was higher than in benign nevi [26]. Upregulated expression of GPR160 was also detected in CD4+CD56+ hematodermic neoplasm, termed blastic natural killer-cell lymphoma [27].
Signal transduction through GPR160 leads to the activation of extracellular signalregulated kinase/cAMP response element-binding protein (ERK/CREB) pathway in rodents [18]. CART induces signal transduction through Gi/o-dependent protein kinase A (PKA)/ERK/CREB phosphorylation in rat nucleus accumbens and hypothalamic paraventricular nucleus [28], but it also stimulates the production of cyclic adenosine monophosphate (cAMP), which indicates the involvement of a Gsα-coupled receptor [29].
ERK is a member of the mitogen-activated protein kinases (MAPK) family that plays a role in cell proliferation, differentiation, migration and apoptosis [30]. ERK1 and ERK2 take part in the activation of cells mostly via growth factors, such as epidermal growth factor (EGF). The binding of EGF to EGF receptors (EGFR) leads to the activation of rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase/ERK (RAS/RAF/MEK/ERK) cascade. Phosphorylation of ERK1 and 2 results in their translocation to the nucleus and further activation of transcriptions factors, eventually leading to changes in specific gene expression. ERK was shown to activate via phosphorylation the 90 kDa ribosomal S6 kinase (p90 RSK ) [31], causing activation of CREB, another transcription factor, which participates in cell cycle regulation [32,33]. RAS and RAF mutations are commonly present in cancer cells and are important factors of tumorigenesis. MEK and ERK mutations are associated with worse prognosis in cancer patients; however, they are rather rare [30,34,35]. The putative roles of CART and GPR160 in the RAF/MEK/ERK pathway and regulation of cell growth and cancer progression are summarized in Figure 2.
The cell cycle is defined as the sequence of events occurring in a cell that enable its division and consists of four stages: G1, S, G2, and M. Its progression depends on at least 48 cell cycle proteins [36] and is regulated by cyclins, cyclin-dependent kinases (CDKs) and a number of kinases and transcription factors. Currently, about 30 different cyclins/cyclinlike proteins and around 20 different CDKs/CDK-like molecules have been identified [37]. Cell cycle checkpoints control the sequence, stability and accuracy of its key events, monitoring, among others, the DNA replication integrity, response to DNA damage, mitotic spindle attachment, chromosome segregation and cell size control [38]. Dysregulation of the cell cycle promotes carcinogenesis by facilitating continuous division [39]. CREB, a potential effector of CART, regulates cell proliferation by modulation of cyclin B, D and proliferating cell nuclear antigen (PCNA) expression in glioma cells [40].
In this qualitative systematic review, we provide a description of the current research focusing on the role of CART in oncologic diseases. We included reports published between 2000 and 2022. After excluding publications that did not meet the inclusion criteria, we analyzed the remaining ones. Then, the reports were categorized based on the type of analyzed material. The role of CART in the development of neoplasms and potential impact on tumor growth is discussed. ylation the 90 kDa ribosomal S6 kinase (p90 RSK ) [31], causing activation of CREB, another transcription factor, which participates in cell cycle regulation [32,33]. RAS and RAF mutations are commonly present in cancer cells and are important factors of tumorigenesis. MEK and ERK mutations are associated with worse prognosis in cancer patients; however, they are rather rare [30,34,35]. The putative roles of CART and GPR160 in the RAF/MEK/ERK pathway and regulation of cell growth and cancer progression are summarized in Figure 2. CART-cocaine-and amphetamine-regulated transcript; CREB-cAMP response element-binding protein; EGF-epidermal growth factor; EGFR-epidermal growth factor receptor; ERK-extracellular signal-regulated kinase; GPR160-G-protein-coupled receptor 160; MEK-mitogen-activated protein kinase; p90RSK-90 kDa ribosomal S6 kinase; PCNA-proliferating cell nuclear antigen; PKA-protein kinase A; RAF-rapidly accelerated fibrosarcoma; RAS-rat sarcoma virus; TF-transcription factor.

Methods
This systematic review of available literature concerning the associations of CART and cancer was performed in accordance with the preferred reporting items for systematic review and meta-analysis (PRISMA) 2020 statement guidelines [41]. The review was registered in OSF Registries with a number osf.io/6kq2g.

Literature Search
A comprehensive literature search was performed by three investigators (MOL, JP and IJ) in the following databases: PubMed, Medline Complete, Scopus and Web of Science, last accessed 8 August 2022. The search was limited to articles in English, published from the year 2000 up to the date of the search, without restrictions to publication type. Our search strategy included the keywords "Cocaine and amphetamine regulated transcript" OR "Cocaine-and amphetamine-regulated transcript" AND "neoplas*" OR "malignan*" OR "growth factor*" OR "cancer*" OR "GPR160", in combinations with and without the abbreviation "CART" as a separate keyword. All results were compared between the investigators and were identical. In the results from databases PubMed, Medline Complete and Web of Science, the number of results did not differ depending on the presence or absence of dashes in the name "cocaine and amphetamine regulated transcript", but in Scopus such a difference occurred, with a larger number of publications with dashes present. Adding the abbreviation "CART" limited the number of results in every database, and all publications found in the search with the abbreviation were also present in searches without it.

Inclusion and Exclusion Criteria
All relevant articles regarding the association of CART with cancer were screened by three reviewers (IJ, MOL and JP) after removing duplicates, based on the titles, abstracts, and full texts. Eligible studies met the following inclusion criteria: (1) the papers were original studies, (2) the used materials were either samples from human patients or cell lines (human or animal), (3) the publication was accessible, (4) it was published in English.
The following types of publications were excluded: (1) reviews, case reports and meeting abstracts, (2) animal studies, (3) studies lacking results relevant to the subject of this paper, (4) publications with duplicated data.
A manual search was also performed in order to identify additional studies in the reference lists of the included publications. Results were described in a narrative manner, without meta-analysis of the data.

Results
Overall, 1182 publications were retrieved (922 in Scopus, 139 in PubMed, 61 in Web of Science and 60 in Medline Complete). After removing duplicates, the number of publications equaled 1033, all of which were screened by title, leaving 41 studies. Nineteen reports were included after the reading of abstracts. Four full-text articles were excluded for the following reasons: the data were duplicated (two papers), the study was a review (one paper), and the paper concentrated only on identifying possible CART receptors (one paper). Five further studies were added after citation screening, leaving a total of nineteen full-text articles included in the review. A flow diagram is presented in Figure 3.    This review comprises 19 publications included after analyzing the full texts. The following cancer types were investigated: breast cancer (five papers), glioma (one paper), and various neuroendocrine tumors (five papers). Data on sample size, patient information and study design are provided in Table 1. Experiments were also performed on cell lines (four papers). Those 15 publications are described in Table 2. The other (four) papers that did not address the relationship between CART and neoplasms but described signaling pathways which were shown to be activated by CART are summarized in Table 3.

CART in Signaling Pathways
Uncontrolled cell proliferation resulting from cell cycle progression is one of the most important mechanisms in carcinogenesis and is promoted by certain signaling pathways, which induce RAF/MEK/ERK and phosphoinositide 3-kinase/3′-phosphoinositide-dependent kinase/protein kinase B (PI3K/PDK/PKB = Akt) pathways. These pathways promote G0 → G1 → S-phase cell cycle progression by induction of cyclin D1 and repression of cyclin-dependent kinase inhibitor 1B (p27 Kip1 ) expression. Akt kinase also causes continuous activation of cyclin/CDK2 complexes and removal of RAF-induced cyclin-dependent kinase inhibitor 1 (p21 Cip1 ) from complexes of cyclin E/CDK2 [42].
On the other hand, CART inhibited the follicle-stimulating hormone (FSH)-stimulated ERK1/2 and Akt signaling in bovine granulosa cells, reducing the kinase activation in a time-dependent manner [45].

CART in Signaling Pathways
Uncontrolled cell proliferation resulting from cell cycle progression is one of the most important mechanisms in carcinogenesis and is promoted by certain signaling pathways, which induce RAF/MEK/ERK and phosphoinositide 3-kinase/3 -phosphoinositidedependent kinase/protein kinase B (PI3K/PDK/PKB = Akt) pathways. These pathways promote G0 → G1 → S-phase cell cycle progression by induction of cyclin D1 and repression of cyclin-dependent kinase inhibitor 1B (p27 Kip1 ) expression. Akt kinase also causes continuous activation of cyclin/CDK2 complexes and removal of RAF-induced cyclin-dependent kinase inhibitor 1 (p21 Cip1 ) from complexes of cyclin E/CDK2 [42].
On the other hand, CART inhibited the follicle-stimulating hormone (FSH)-stimulated ERK1/2 and Akt signaling in bovine granulosa cells, reducing the kinase activation in a time-dependent manner [45].

CART in Cell Proliferation
The effect of CART on cell proliferation was confirmed by increased cyclin D1 protein expression in CART-treated GLUTag cells (murine enteroendocrine cancer cell line) [46]. As mentioned by Sathanoori et al. [43], the exposure to CART modulates cell proliferation and survival; it induces the phosphorylation of p44/42 MAPK at Thr-202/Tyr-204, FoxO1 at Ser-256, p90 RSK (a downstream target of p44/42 MAPK) at Ser-380, and the phosphorylation of CREB and IRS proteins. CART exposure also increases cAMP levels, as shown in β-cells. CART-mediated proliferation in INS-1(832/13) cells was prevented by the addition of pharmacological inhibitors of PKA, Akt and MEK1. Thus, CART, via enhanced cAMP production, seems to regulate downstream effectors, including PKA and MAPK [43]. Both effects promote cellular survival and proliferation since MAPK p44/42 signaling inhibition enhances proteasome inhibitor-mediated apoptosis [47], while cAMP enhances the neuregulin-dependent proliferation of Schwann cells [48].
The data presented above indicate that CART may promote tumorigenesis via multiple pathways. Correspondingly, CART levels are also elevated in various cancer types, both in patients' plasma and in the tumor cells themselves, vide below.

CART Activity in Breast Cancer
Five publications discussed the association of CART and different types of breast cancer.
It has been shown that CARTPT mRNA is expressed in cytological specimens acquired during duct-washing cytology (DWC) derived from solid papillary carcinomas (SPC), a subtype of ductal carcinoma in situ (DCIS), and in intraductal papillomas. Jikuzono et al. [49] chose CARTPT and breast cancer-associated transcript 54 (BRCAT54) mRNA as markers of quality and quantity of RNA extracted from DWC in the diagnosis of DCIS. DWC samples (12 of 37) were characterized with an RNA integrity of equal or more than 6, representing moderate to high quality. Then, five samples (three malignant, two benign) of DCIS with high RNA yields were elected for quantitative RT-PCR analysis of CARTPT and BRCAT54. mRNA of CARTPT and BRCAT54 were detectable in all the samples subjected to quantitative RT-PCR, as well as the mRNA of HPRT-1 (hypoxanthine phosphoribosyl transferase 1, a housekeeping gene), although the CARTPT/HPRT-1 ratio varied from 0.1 to 14.2 in the few samples measured (values from 0.1, 0.1, 0.2, 2.8 to 14.2). Since the measured markers were detected in all samples, RNA from DWC CARTPT mRNA was implicated as a potential biomarker of DCIS [49].
Lu et al. [50] compared two histological types of breast cancer: mucinous adenocarcinoma (MC) and invasive ductal carcinoma. Data concerning clinico-pathological features of 186 497 patients were obtained from the Surveillance, Epidemiology, and End Results Program (SEER) 18 database. The mean age of MC patients was higher, with a lower tumor N and G stage value. The proportion of MC cases with positive estrogen receptor (ER) and progesterone receptor (PR) was higher, but it was lower in the case of positive human epidermal growth factor receptor 2 (HER2). Mucinous adenocarcinoma patients received chemotherapy or radiotherapy, and they had a mastectomy less often. Genomic and transcriptomic analysis of 801 cases of IDC or mucinous adenocarcinoma was conducted based on data from The Cancer Genome Atlas (TCGA) database. CARTPT expression was found to be upregulated in mucinous adenocarcinoma cases together with MUC2 and TFF1 genes [50].
In another article by Brennan et al. [51] the influence of CART on ER-positive, lymph node-negative breast cancer was investigated, together with the possibility of using CART in the diagnosis of this type of tumor. Initially, immunochemistry performed on 10 singlepatient tissue microarrays showed that CART protein was expressed in 20% of cases.
Expression was restricted to tumor cells and was not evident in normal breast epithelium. Then, after transfection of MCF-7 breast cancer cells with a CMV-CART construct (CART cDNA with a pCMV6-XL5 plasmid vector), it was observed that CART stimulates the expression of CART in other cells in an autocrine and paracrine way. CART was also found to be a stimulant of the ligand-independent activation of ER alpha by MAPKmediated phosphorylation of ER alpha at S188. Finally, in three independent cohorts of ER-positive, lymph node-negative breast cancer, the prognostic factor of CART was assessed. It was shown that high CART expression is associated with decreased overall survival in lymph-node-negative cancer. Moreover, CART is a predictor of the outcome in tamoxifen-treated patients. Increased CART expression was correlated with an impaired response to tamoxifen. CART also protects the breast cancer cell line from tamoxifenmediated death [51].
In the study by Slattery et al. [52], several energy homeostasis genes were evaluated to find associations of body size measures and breast cancer risk in 3592 cases of breast cancer and 4182 healthy women of either Hispanic/Native American or non-Hispanic white origin. The study also included analysis of five selected single nucleotide polymorphisms (SNPs) of the CARTPT gene. The adaptive rank truncated product (ARTP) method revealed that CARTPT was associated with the risk of pre-menopausal breast cancer in all women (P ARTP = 0.014) and in the group of low Indigenous American (IA) ancestry (P ARTP = 0.015). Interestingly, a similar correlation was found for ghrelin prepropeptide in pre-menopausal women of low IA ancestry (P ARTP = 0.007) [52].
A similar study was conducted by Rodríguez-Valentín et al. [53]. The aim of their report was to check whether chosen energy homeostasis genes (including the CARTPT gene) may modify the association between serum levels of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) and the risk of breast cancer (BC). The population of the study was limited to pre-menopausal women and included 265 cases of breast cancer and 437 healthy women. DNA was extracted from blood samples, and the serum levels of IGF-1 and IGFB-3 were measured. The participants were divided into tertiles based on IGFBP-3 serum levels. No influence of the CARTPT gene on the association between IGF-1 serum level and BC risk was found. There was no association between IGFBP-3 serum level and CARTPT rs3846659 SNP. However, CARTPT rs3846659 presented to be a significant modifying factor for the association between IGFBP-3 and the risk of BC. In women with dominant homozygotes in the CARTPT SNP rs3846659-that is, with the GG genotype-the risk of developing breast cancer was shown to be increased when comparing the highest and the lowest tertile of IGFBP-3 serum levels. No effect was observed in the presence of the minor allele C. These observations show that the CARTPT gene polymorphism modifies the association between IGFBP-3 and BC risk [53].

CART Activity in Neuroendocrine Tumors
Connections between CART and neuroendocrine tumors were investigated in four articles.
Ramachandran et al. [54] compared the usefulness of CART, chromogranin A (CgA) and chromogranin B (CgB) as biomarkers of various NETs, facilitating the diagnosis and judgment of disease progression. The investigated cancer types included pancreatic, gut, phaeochromocytoma/paraganglioma, nongastroenteropancreatic and unknown primary tumors. The levels of all three peptides were measured in the plasma of 481 confirmed NET patients and compared with control samples from 40 healthy volunteers. The results showed that CART is more useful than both CgA and CgB in the detection of stable and progressive phaeochromocytomas/paragangliomas. This could make CART a surveillance marker for patients with these types of NETs. It was also confirmed that CART is a reliable marker for the identification of progressive pancreatic NETs [54].
Bech et al. [55] investigated a range of neuroendocrine malignancies: gastric, midgut, hindgut, pancreatic, unknown primary, pulmonary, thymic, paraganglionic, ovarian, and renal. CART levels were studied as a tumor marker of neuroendocrine malignancy in the plasma samples from 131 patients. CART levels were also measured in twenty-seven patients after removal of NETs, six patients with newly diagnosed pituitary tumors, thirty-four with various non-neuroendocrine tumors, 153 prostate cancer patients and seventeen patients with renal impairment, in order to investigate whether the measurement of CART levels facilitates identification of nonfunctioning NETs. The control group consisted of 192 patients with other conditions. The plasma CART levels of the vast majority (95%) of patients with progressive pancreatic NETs were increased, with the mean plasma CART levels significantly higher (p = 0.035) in those suffering from progressive disease in comparison to patients with all types of stable NETs. When measuring CART and CgA together, their combined sensitivity for discovering neuroendocrine malignancies was higher than the combined sensitivity of CgA and CgB. This shows that CART is a better biomarker than CgB [55].
Landerholm et al. [56] focused on establishing whether CART is expressed in various types of neuroendocrine tumors and, if so, examined CART-expressing cells. Specimens from gastric, ileal, rectal carcinoids, endocrine pancreatic tumors and medullary thyroid carcinomas were used for histopathological examinations and immunohistochemistry. CART-IR (CART immunoreactivity) was found in 80% of all examined NETS; however, its intensity varied between cells. Origin of the neoplasm did not influence CART expression. Either no differences were observed between primary tumors and metastases, or no coexpression of CART and Ki-67 (a cell proliferation marker) was detected [56].
In another study, Landerholm et al. [46] evaluated the role of CART in small bowel carcinoid tumors in a group of 97 patients by histopathological examination and immunohistochemistry. CART expression and higher expression patterns were associated with worse survival. However, CART expression did not correlate with the patients age, disease stage, tumor grade or any manifestation of disease. Next, the effect of CART on tumor cell survival was assessed using GLUTag and HCT-116 cells. CART increased cell viability in both cell lines. In GLUTag cells, an increased cyclin D1 protein expression was observed following CART-treatment [46]. Since cyclin D1 promotes cell cycle progression, CART may stimulate cell division and thus trigger carcinogenesis.

CART Activity in Other Neoplasms
Two articles investigated the role of the CARTPT gene in the development of other cancer types.
In the publication by Wang et al. [57] the correlation between variations of the CARTPT gene and susceptibility to glioma in the Chinese population was evaluated. The stage I study included 400 patients with a glioma and 400 healthy controls. Four SNPs were selected, and their associations with glioma risk were assessed. Two variants were associated with an increased glioma susceptibility-rs2239670 and rs11575893. Variants rs3846659 and rs6894772 did not display any correlations with the risk of developing glioma. These results were further validated based on 800 glioma cases and 800 healthy controls. Finally, a statistically significant association between rs2239670 (OR = 1.27; 95% CI = 1.10-1.46; p = 0.001) and rs11575893 (OR = 1.25; 95% CI = 1.09-1.45; p = 0.002) with a higher glioma risk was confirmed in the pooled patient and control groups [57].
Zhou et al. [58] used datasets from TCGA to explore the tumor microenvironmentrelated genes in cases of stomach adenocarcinoma. The CARTPT gene was associated with survival rate-high gene expression correlated with worse survival probability. This was confirmed using data from the Gene Expression Omnibus database [58].

CART Activity in Cancer Cell Lines
Four publications described the influence of CART on cancer cell lines-rat adrenal pheochromocytoma-PC12 [59,60], rat insulinoma-INS-1(832/13) [43] and murine pituitary tumor-AtT-20 [44]. (1) CART is expressed in primary and metastatic breast cancer.  (1) Association between the CARTPT gene and the risk of breast cancer in a group of pre-menopausal women with low Indigenous American ancestry and in the group of all pre-menopausal women. [52] Breast cancer Analyses of modifying effect of 10 energy homeostasis genes (including CARTPT) on the association between IGF-1 and IGFBP-3 serum levels and the risk of BC.
(1) No effect of CARTPT on the association of IGF-1 serum level and BC risk. [53] Neuroendocrine tumors (NETs): To evaluate the comparative and combined utility of CART, CgA and CgB for diagnosis and monitoring of different NET subtypes. (1) CART-marker for the identification of progressive pancreatic NETs; superior to CgA and CgB in detecting stable and progressive PCC or PGLs. (2) CART plasma levels decreased after resection of tumors, remained low in all patients in remission, but increased when progression of the disease occurred. (3) The measurement of CART did not improve diagnosis in gut, nonGEP NETs, and NETs with an unknown primary origin in comparison with combined CgA and CgB measurement.
[54] To investigate the potential role of plasma CART levels as a biomarker of neuroendocrine malignancies.
• Twelve volunteers-analysis of fasting effect on plasma CART levels. • A total of 131 patients with neuroendocrine tumors: fifty-one midgut, thirty-eight pancreatic, five hindgut, five pulmonary, two thymic, two paraganglionic, two gastric, one ovarian, one renal, twenty-four of unknown origin. Sixty-eight patients with stable, sixty-three with progressive illness. • Measurement of plasma CgA, CgB and CART immunoreactivity levels (gel chromatography and reverse-phase fast protein liquid chromatography).
Maletínská et al. [59] investigated the binding of iodinated CART peptide ( 125 I-CART 61-102 ) to PC12 cells. Those were both non-differentiated as well as treated with nerve growth factor (NGF) and developed into neuronal phenotype. It was shown that 125 I-CART 61-102 binds specifically to both investigated cell types; however, the binding capacity of differentiated cells was higher than of those non-differentiated. CART was bound to intact cells, and isolated membranes as well. The results suggested the presence of CART receptors (not known at that time) on PC12 cells and cells derived from them [59].
Indeed, Lin et al. [60] later investigated the pharmacological profile of CART binding sites on the PC12 cell line and the association between increased CART binding and CARTactivated signaling in PC12 cells after their neuronal differentiation. Before differentiation, CART's influence was small but significant, causing an increase in ERK phosphorylation. The addition of CART 61-102 to NGF-treated cells produced a much more considerable increase in phospho-ERK (p-ERK), demonstrating that CART-activated signaling intensifies with differentiation. Similarly, an increase in CART binding capacity occurs with differentiation of PC12 cells. A GPCR coupled to Gi/o can mediate CART signaling in these cells [60].
In the publication by Sathanoori et al. [43], the effect of exogenous CART 55-102 on βcell viability was judged. The signaling mechanisms of this peptide were also investigated. Studies on the INS-1(832/13) clonal β-cell culture and isolated rat islets showed that CART decreased glucotoxicity-induced apoptosis in the INS-1(832/13) cells and β-cell apoptosis in the rat islets (by 63% and 66%, respectively) [43].
Lakatos et al. [44] revealed that CART 55-102 activates ERK1 and 2 in a time-and dosedependent manner in AtT20 cells, and that this effect is absent in the presence of U0126, a highly selective MEK1 and 2 inhibitor, demonstrating an involvement of these kinases in the signaling pathway. Since pertussis toxin, a Gi and Go signaling inhibitor, hindered p-ERK formation, the involved receptor is likely to be a Gi/o coupled GPCR. Genistein, an inhibitor of receptor tyrosine kinase signaling, did not exert such effects [44].

Conclusions
The results of the studies included in this systematic review suggest that CART could be a potential valuable diagnostic biomarker in various types of oncological diseases, which may lead to the improvement of early cancer detection. There is an increasing amount of evidence showing the potential role of CART in the modulation of neoplastic processes. Therefore, further experimental and clinical research is needed to clarify the role of CART in cancer pathogenesis. Identifying precise mechanisms of CART action may result in the development of new therapeutic approaches and the design of novel antineoplastic agents.